1. Field of the Invention
The present invention relates generally to the detection of microbial contamination in ultra-pure water systems, but more specifically to the detection and/or identification of microbial biofilms which are predisposed to form on surfaces of the ultra-pure water system where the sparse nutrients therein tend to accumulate.
2. Description of the Prior Art
The recovery of bacteria in high-quality water used for drinking and in ultra-pure water systems used to process integrated circuits or other electronic devices is a major problem. In ultra-pure water systems, any type of contamination is a major problem. The contamination of integrated circuits with microorganisms is an especially serious problem as these organisms have the potential to proliferate and destroy the function of the circuits. The organisms that colonize the ultra-pure water systems are initially bound or attached to the surfaces of the containment systems as biofilms because the surfaces are the sources of what little nutrient are available in these environments. A source of the bound organisms is from breakthrough or regrowth from other organisms that were insufficiently treated at the purification center, and, accordingly, recovered from the disinfectant treatment or avoided the treatment. Another source of bound organisms is from true breakdown from contamination by back siphonage or cross connections in the ultra-pure water systems.
The contamination problem is further complicated by the strong tendency of organisms in these oligotrophic (nutrient-poor) environments to attach to particles or surfaces with the formation of biofilms. These biofilms are anchored to the surfaces by exopolymer polysaccharides (EPS) which make them extremely difficult to treat and to dislodge quantitatively for prior art culturable methods by which they are routinely detected.
The prior art evaluation methods that rely on the culturing of these organisms suffer several serious deficiencies. Organisms from these extreme environments, i.e., high-quality or ultra-pure water systems, are notoriously difficult to culture so that standard plate count techniques will not give an accurate estimate. The problem of viable but not culturable bacteria is a well known and accepted concept in public health microbiology. For example, the cholera causing organism Vibrio cholera when starved forms minicells that are often not culturable, but readily initiate the disease when the water is consumed. Not only do the microorganisms not grow out readily on attempted isolation, but the growth, particularly, of autotrophic organisms found in very low nutrient water take several days to weeks to grow to detectable colonies. This delay is clearly not acceptable in ultra-pure water systems where continual control is essential. Many of the prior art microscopic techniques are not very sensitive. It often requires at least 10.sup.5 organisms/ml for reliable detection of contamination. Concentration by centrifugation or filtration with vital staining and microscopic examination requires that the water system be sampled, thereby creating the potential for additional contamination. These methods are also time consuming, and require trained operators for detection and identification. Moreover, the type(s) of bacteria involved in the contamination are not usually readily determined with the microscopic examination. It is possible to identify specific microbes using fluorescent labeled probes. This requires, however, that the organisms commonly contaminating the system be well known so that antibodies or gene probes can be developed.
A well known prior art method of enumerating bacteria is to recover them from water by filtration or from containment surfaces by mechanical or physical (ultrasonic) means. The bacteria are then cultured in pour plates of various nutrient media. There are various prior art methods that allow for the recovery of damaged bacteria so that they will eventually grow and form visible colonies in an assay system. If any of these techniques are to be accurate, two main features must be established. First, the bacteria must be quantitatively recovered from the biofilm or the filter so they can be plated in the nutrient media used for the colony formation. Second, the nutrient conditions must be able to grow every organism from a single cell to a colony of over 10.sup.6-9 cells. It is known in the prior art from experiments in seawater that the number of bacteria that can be counted directly on a filter after staining versus the number that can be determined by plate counts is between 10.sup.2 to 10.sup.4 times less. This problem is more clearly demonstrated by experience in soils where the attachment problem complicates the culturability problem. Here, often considerably less than 0.1% of detectable organisms can be cultured.
The problem of culturability is even worse in the high-quality or ultra-pure water systems because the bacteria in the "regrowth" condition which have been injured sublethally by disinfection treatment account for most of the increased bacterial contamination. The problem of infectious bacteria that are "infectious but not culturable" has recently become known in the prior art. The disinfection-injured bacteria in the ultra-pure water systems particularly those attached to particles or containment surfaces are known to be very difficult to culture. Accordingly these ultra-pure water systems are vulnerable to distribution of contaminated water having organisms that will colonize the surfaces of the distribution system with the concomitant health and property problems.
The prior art, as indicated hereinabove, teach some advances in the detection/identification of microbial contamination in high-quality or ultra-pure water systems. Insofar as can be determined, however, no prior art apparatus or method incorporates all of the features and advantages of the present invention.